KR20090112955A - Food comprising Corchorus olitorius L. extract and its fractions having anti-inflammation activity - Google Patents

Abstract

PURPOSE: A food containing Corchorus olitorius L. extract is provided to increase total polyphenol and flavonoid content at the same time exhibiting high DPPH and ABTS removal effect. CONSTITUTION: A food containing Corchorus olitorius L. extract is produced using a solvent, standard product, and experiment equipment. The solvent for use in extracting and fractioning a sample is methanol, n-hexane, chloroform, ethylacetate, or n-butanol. The standard product for use in HPLC is quercetin, chlorogenic acid, quercetin-3-glucoside, or quercetin-3-galactoside. DMEM, antibiotic, Fetal bovine serum(FBS) are used for cell cultivation. A reagent for use in RNA extraction is Trizol solution Molecular Research Center, or 1-bromo-3-chloro-propane.

Description

Food comprising Corchorus olitorius L. extract and its fractions having anti-inflammation activity

The present invention relates to a food containing a molochia extract, and more particularly to a food containing a molochia extract having anti-inflammatory activity.

Recently, the situation of disease has changed due to economic growth and dietary change, and chronic degenerative diseases caused by overnourishment or malnutrition such as arteriosclerosis, obesity, diabetes, and cancer are continuously increasing. One of the main factors of this chronic disease is reactive oxygen species (ROS). The presence of ROS in the body is eliminated by the operation of the body's elimination system or reduction mechanism.However, if the amount of production is excessive compared to the amount removed, oxidative stress caused by the ROS in the body can damage DNA, protein, lipid molecules, Peroxidation can lead to cell damage that can lead to a variety of diseases.

  In general, atherosclerosis is a disease in which the artery wall becomes thick and hard, resulting in loss of elasticity, and is commonly referred to as atherosclerosis, and is defined as a disease in which fatty plaques accumulate inside the endocardium and the media. Atherosclerosis is also reported to be a chronic inflammatory disease, not just a disease associated with the aging process. Factors that induce atherosclerosis include elevated blood cholesterol levels, hypertension, diabetes, obesity, stress, and smoking. Especially, oxidation of low density lipoprotein (LDL) is important for the formation and progression of early atherosclerotic lesions. It is known to play a role.

  These risk factors impair the function of vascular endothelial cells to move LDL in the blood to endothelial tissue, which is oxidized by endothelial cells, smooth muscle cells, macrophages and the like. Oxidized LDL (oxidized LDL) is a vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (IMC-1), and monocyte chemoattractant protein -1) by expressing cytokines NFκB, IL-1, TNF-α and growth factors MCSF and PDGF, help to induce monocytes in the blood to continue arteriosclerosis.

 Oxidative stress regulates gene expression in specific cells that cause degenerative diseases as well as promote inflammatory responses and cytotoxicity. Among them, NFκB activates IκB kinase by chemokines such as ROS, TNF-α, and IL-1β, and then, after phosphorylation and a series of degradation processes, isolates IκB and transfers them to the nucleus of NFκB. NFκB is known to promote the expression of genes such as cytokine, iNOS, COX-2, VCAM-1, ICAM-1, MCP-1, and E-selectin, which are activated and move to the nucleus to induce an inflammatory response. Therefore, reducing NFκB activity and expression of cell adhesion molecules can prevent various inflammation-related diseases.

On the other hand, Corchorus olitorius L. is a year-old greenish-yellow vegetable of the barberry family , originating from the Mediterranean coast of Egypt, originating from the balanced balance of protein, crude fiber, ash, vitamins and minerals, especially 22.8% mucilage, and β-carotene and lutein are also abundant. In addition, Molochia has been reported to have physiological activities such as antioxidant activity and cholesterol lowering effect by phenolic substances.

In one embodiment of the present invention to provide a food comprising a molochia extract having anti-inflammatory activity.

In one embodiment of the present invention in particular to provide a food comprising a fraction of the molochia extract excellent in anti-inflammatory activity.

In one embodiment of the present invention to provide a food containing a molochia extract and fractions having excellent inhibitory ability to oxidative and inflammatory reactions.

In one embodiment of the present invention provides a food having an anti-inflammatory activity, including the Molochia methanol extract.

One embodiment of the present invention also provides a food having anti-inflammatory activity comprising an ethyl acetate fraction of the Molochia methanol extract.

One embodiment of the present invention also provides a food having anti-inflammatory activity containing quercetin-3-galactoside derived from molokia.

Food according to the embodiments of the present invention may be to suppress the formation _{NO (nitric oxide), PGE 2} (Prostagladin E 2), and TNF-α (Tumor neroses factor- 1).

Food according to embodiments of the present invention, may be to inhibit the mRNA gene expression of MCP-1 (Monocyte chemotactic protein-1), iNOS (Inducible NOS), COX-2 (Cyclooxygenase-2).

Food according to embodiments of the present invention may be to inhibit the protein expression of COX-2 (Cyclooxygenase-2), NFκB (Nuclear fator κB), IL-1β (Interleukine-1β).

According to one embodiment of the present invention, it was confirmed that the methanol extract, especially ethyl acetate fraction of Molochia, has high total polyphenol content, high total flavonoid content, high DPPH and ABTS scavenging activity, and has excellent antioxidant ability.

In addition, according to another embodiment of the present invention using a RAW 264.7 cell line that caused oxidative stress, as a result of investigating the effects of the extract and fractions on the oxidation and inflammatory reaction, 50 ~ 250 μg / viability, NO production, PGE ₂ at mL concentration It was confirmed that TNF-α production ability was inhibited, in particular, cell viability, NO production, PGE ₂ , at a concentration of 100 μg / mL of the molokia ethyl acetate fraction. It was confirmed that the TNF-α generating ability was most suppressed. In addition, mRNA expression of MCP-1, iNOS, and COX-2 was inhibited at the concentration of 100 μg / mL of ethyl acetate fraction, and protein expression of NFκB and IL-1β was also inhibited.

As a result, it was confirmed that the ethyl acetate fraction of the Molokia extract, especially the Molokia methanol extract, had excellent antioxidant activity and had anti-inflammatory activity. Therefore, Molokia extracts and fractions will be said to be excellent food materials with anti-inflammatory activity.

Inflammatory responses are promoted by oxidative stress in the body, which triggers or worsens the inflammatory response by increasing the gene expression of specific cells that cause not only cell death but also degenerative diseases. Among them, NFκB is a factor that regulates cell proliferation and growth, inflammatory response, and cell adhesion.It is mainly in complex with inhibitory protein IκB and is present in an inactivated state, and it is a factor that promotes division, inflammation, cytokines, ultraviolet rays, ionization, ROS When stimulated by bacterial LPS, IκB is rapidly phosphorylated and degraded. At this time, NFκB isolated from IκB is activated and moved to the nucleus to regulate several genes involved in the initial inflammatory response. The representative ones are inducible NOS (iNOS) and cyclooxigenase-2 (COX-2).

Radical nitric oxide (NO ^-), one of the species is produced by the nitric oxide synthase (NOS) from the guanidine nitrogen and oxygen in the L-arginine, the enzyme, the endothelial NOS (eNOS), neuronal NOS (nNOS), iNOS 3 There is an isomer of eggplant. Among these, iNOS mRNA is expressed in both early and advanced lesions of arteriosclerosis in animals or humans. In particular, iNOS mRNA and oxLDL are highly expressed in abundant phagocytes, and iNOS is involved in atherosclerosis.

In addition, COX-2 is one of the major regulatory enzymes of prostaglandin biosynthesis from arachidonic acid, which is hardly detected in normal tissues and is easily induced by oncogenes, growth factors, and cytokines. In particular, inhibition of COX-2 can inhibit inflammation, so COX-2 is a major regulator of inflammation.

Antioxidants are substances that stabilize or inactivate free radicals before attacking cells, and mainly mean vitamin C, vitamin E, β-carotene, and polyphenols. Among them, polyphenol compounds have strong antioxidant activity, have been reported to inhibit COX (cyclooxigenase) and reduce NFκB activity.

Inhibition of NFκB activity occurs by inhibiting the release of inflammatory mediators in vitro and in vivo . In addition, the polyphenolic compound inhibits the activity of AP-1, a nuclear factor, regulates the expression of COX-2, increases the activity of HDL-related paraxonase-1 (PON-1) and antioxidants in the blood, and the human aortic epithelium. Reduction of VEGF expression in cells inhibits the expression of IFN-r and IL-4 genes in monocytes and macrophages in normal peripheral blood. Quercetin inhibits the production of LPS-induced cytokines such as IL-1β, TNF-α by inhibiting NFκB activity, and polyphenols of curcumin, resveratrol, theophylline, flavonols, and green tea inhibit NFκB activity, It inhibited the expression of early inflammatory genes such as IL-8, TNF-α and iNOS, and showed anti-inflammatory activity by inhibiting COX and lipoxygenase pathways (see FIG. 1).

Recent intake of polyphenols has been shown to reduce coronary heart disease or ischemic shock mortality. Natural plant extracts, which have been used traditionally, are rich in polyphenolic compounds, which can inhibit the progression of arteriosclerosis in animal models. Polyphenolic compounds of grape seed, wine and green tea prevent the development of atherosclerosis in animal models and delay the formation of plaque in the early atherosclerotic lesions, especially in the polyphenol compounds quercetin, catechin, caffeic acid and resveratrol. Inflammatory effect was confirmed.

Many plants have polyphenolic compounds, including phenolic acid and flavonoids. Flavonoids among polyphenols are compounds that are widely present in plant systems, and are mainly contained in vegetables, fruits, and wines. They are classified into more than 5,000 different flavonoids such as flavonol, flavone, isoflavonol, flavanol, anthocianin and proanthocianidin (Fig. 2).

Flavonols are the most common flavonoids in foods, including quercetin, kaempferol and myricetin. In particular, quercetin is a major phenolic component of plants and is the most important quantity of dietary flavonoids. Most quercetin in foods is in the form of glycosides bound to sugar, and there are free flavonoids that some sugars do not bind to, called aglycone. Aglycone is absorbed by the small intestine mucosa, but most of the flavonoids in the diet are glycosides and have a large molecular weight, so they are hardly absorbed by the small intestine mucosa.

Flavonoids are known to have physiological activities such as antioxidants, anti-inflammatory reactions and anticancer. Green vegetables, tomatoes, and melons prevent colon and rectal cancer, and people who eat less fruit or vegetables reported a 17 percent higher mortality rate from heart disease than those who consumed more flavonoid-rich fruits or vegetables. Flavonoids are typically isolated from natural products due to structural features such as O-dihydroxyl group of β-ring, 4-oxo group of C ring, conjugated double bond of 2,3 position, and 3,5-hydroxyl group of A and C ring. Known as an antioxidant. Flavonoids also form stable metal-ion complexes with Fe ²⁺ and Cu ²⁺ , which eliminate chain-initiating radicals on the cell membrane surface and promote free radical damage, and free radicals such as superoxide anione, hydroxyl radical and peroxy radical. It is known to remove it directly.

Flavonoids, in particular, are mostly hydrophilic and do not bind to LDL molecules and act as a free radical chelating agent or chelating agent to protect vitamin E, carotene and lycophen from LDL fragments, and to maintain paraoxonase activity in the blood that can hydrolyze lipid peroxides. Inhibits LDL oxidation. In addition, several in vitro studies have reported that polyphenols protect LDL from oxidation and may reduce oxidation of LDL even when ingested with flavonoid-rich natural products.

In one embodiment of the present invention, in order to determine the anti-inflammatory effects of Molochia, first, the Molokia extract and fractions were prepared to measure the antioxidant effect in vitro , the activity of NO, PGE ₂ and TNF-α of inflammatory reaction, and oxidation Expression patterns of VCAM-1, ICAM-1, and MCP-1, which are required for adhesion to monocytes by LDL, were identified. We also compared the anti-inflammatory effects of quercertin, chlorogenic acid, quercetin-3-galactoside and quercetin-3-glucoside.

This will be described in detail below.

1. Materials and Methods

1.1. Reagents and Instruments

1.1.1. reagent

The solvents used for sampling and fractionation, methanol, n- hexane, chloroform, ethylacetate and n- butanol, were prepared using JTBaker (Mallinckrodt Baker Inc., Philipsburg, USA). As quercetin, chlorogenic acid, quercetin-3-glucoside, and quercetin-3-galactoside, Sigma (St. Louis. Mo., USA) was used. DMEM, antibiotic, Fetal bovine serum (FBS) and Trypsin-EDTA used for cell culture were purchased from Gibco BRL (Rockville, USA). 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide (MTT) measured for cytotoxicity was purchased from Amresco (Ohio, USA). Primer used in the PCR experiment was from Bioneer (Daejeon, Korea), and the reagent used for RNA extraction was Trizol solution Molecular Research Center (Ohio, USA), and 1-bromo-3-chloro-propane was Sigma (St. Louis.Mo. , USA) and RT-PCR were measured by Takara kit for Takara (Shiga, Japan) and DNA ladder for Promega (Madison, WI, USA). And trizma base (Tris-Cl), ethylenedinitro tetraacetic acid disodium salt (EDTA-2Na), triton X-100, N, N, N ', N'-tetra-methylethylene diamine (TEMED), acrylamide , sodium dodecyl sulfate (SDS), ammonium persulfate (APS), tween-20, and bicinchoninic acid kit were all purchased from Sigma (St. Louis.Mo., USA), and ECL Detection kits were obtained from Amersham (Buckinghamshire, England), And immobilon-P transfer membrane was purchased from Millipore (Billerica Massachucetts, USA), NFκB (p65), IL-1β, iNOS, COX-2 from Santacruz biotechnology (California, USA).

1.1.2 Appliance

Important instruments used in the experiments were rotary vacuum evaporator (Buchi, R-3000, Germany), UV / visible spectrophotometer (Kontron, Uvikon 922, Italy), incubator (Forma Scientific, Model 3154, USA), HPLC (Shimadzu LC-10A prominence , Shimadzu, Japan), PCR machine (Bioneer, Mygenie 96, Korea), Mini-Protein electrophoresis system (BioRad Co., USA), microplate spectrophotometer (Molecular Devices, Spectra max 340PC, USA), microplate shaker (Finepcr, Finemixer SH2000 , Korea), ultracentrifuge (Hitachi, Model 695-7, Japan), cryotome (Leica CM 1850, Germany), micro haematocrit centrifuge (Hanshin HHC-24, Korea) were used in this experiment.

1.2 Experimental Materials

Mollocia ( Corchorus olitorius L.) was dried at 50 ℃ for 12 hours at the end of the leaves collected in late August 2006 and then used to buy the powder pulverized into 100 mesh village Yeoju, Gyeongju-do. 10 times 80% methanol (v / v) was added to the powder sample, and the extraction was repeated three times. The extract was then filtered twice using filter paper, concentrated in a vacuum condenser, lyophilized and used as a methanol extract. Methanol extract was further fractionated with n- hexane, chloroform, ethylacetate, n- butanol and water, concentrated and dried to prepare a fraction, which was used as a sample.

1.3 Antioxidant Effects of Molokia

1.3.1 Total Polyphenol Content

Total polyphenol content was measured by applying the Folin-Denis method. Dissolve 1 mg of each methanol extract sample in 1 mL of distilled water, add 2 mL of diluted Folin reagent to 2 mL of 10-fold dilution, mix, leave for 3 minutes, add 2 mL of 10% Na ₂ CO ₃ , and add 1 After standing for some time, the absorbance was measured at 700 nm using a UV / visible spectrophotometer. The total polyphenolic compound was determined from the standard curve prepared using tannic acid.

1.3.2 Total Flavonoid Content

Total flavonoid content was determined by Nieva Moreno et al. (Nivea, MM; Sampietro, AR; Vattuone, MA Comparison of the free radical-scavenging activity of propolis from several regions of Argentina.J. Ethnopharmacol. 2000 , 71 , 109-114.) Measured by Dilute each sample solution 10 times with 10% ethanol, take 100 μL, mix it with 4.3 mL of 80% ethanol containing 10% aluminum nitrate and 1 μM potassium acetate, and leave for 40 minutes at room temperature. Measure the absorbance at 415 nm. It was. Total flavonoid content was determined from the standard curve prepared using quercetin.

1.3.3 Scavenging Activity of DPPH Radicals

The scavenging activity of DPPH radicals was determined by Blois (Blois, MS Antioxidant determinations by the use of a stable free radical. J. Agric. Food Chem. 1977 , 25 , 103-107.). Was measured. Each extract was dissolved in 99% methanol by concentration, 800 μL was taken and mixed with 200 μL of a 0.15 mM DPPH solution dissolved in methanol, and then absorbance was measured at 517 nm after 30 minutes. The free radical scavenging activity of each sample extract was expressed by the RC ₅₀ value, which is the concentration of the sample required to reduce the absorbance of the control group without the sample to 1/2.

1.3.4 ABTS radical scavenging activity

Antioxidant activity using 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical was measured by ABTS + ˙ cation decolourisation assay (Re, R .; Pellegrini, N .; Proteggente, A .; Pannala). , A .; Yang, M .; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay.Free Radical Biol. Med. 1999 , 26 , 1231-1237. 7 mM ABTS and 2.45 mM potassium persulfate were mixed at the final concentration and left for 24 hours in the dark at room temperature to form ABTS + ˙. The absorbance value was 0.70 (± 0.02) at 732 nm (PBS, pH 7.4). Dilution). 10 μL of sample was added to 990 μL of the diluted solution, and the absorbance was measured after standing for exactly 1 minute. The free radical scavenging activity of each sample extract was expressed by the RC ₅₀ value, which is the concentration of the sample required to reduce the absorbance of the control group without the sample to 1/2.

1.3.5 Polyphenol Compound Analysis

The high performance liquid chromatograph used for the analysis used Shimadzu's HPLC 10-A. ODS-HG5 (4 mm, 4.6 i.d. x 150 mm) was used as a column to separate each component, and it was mounted in a column oven maintained at 40 ° C to be reproducibly separated at constant temperature. The mobile phase used two buffers. Buffer A was distilled water containing 2% acetic acid, and buffer B was used 50% acetonitrile. Gradient conditions for separation are shown in Table 1 below, and analyzed at a flow rate of 0.9 mL / min. Detection was measured at a wavelength of 295 nm using a UV-VIS detector (SPD-20A).

Time (min) Buffer A (%) Buffer B (%) 0 100 0 0.3 90 10 50 55 45 70 0 100 79 10 90

1.4 NO production and anti-inflammatory effects

1.4.1 Cell Culture and Treatment

RAW 264.7 cells, a Murine macrophage cell line, were distributed from Korean Cell Line Bank (KCLB), and 5% CO ₂ was added using DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin). Passage was performed 2-3 times a week in a 37 ℃ incubator.

Cells were inoculated in D60 culture dish (1 dish10 ⁶ cells / dish) and 24 well plate (1 ㅧ 10 ⁵ cells / well), adhered to the sample, incubated for 24 hours after treatment by concentration, and lipopolysaccharide (LPS) 1 After incubation for 24 hours by adding μg / mL was used in the experiment. The experimental group without LPS was used as a negative control, and the LPS treated as a positive control.

1.4.2 Cell viability measurement

The NO produced by the Cytokine in order to observe the effect on RAW 264.7 cell Green, etc. (Green, LM; Reade, JL ; Ware, CF Rapid colometric assay for cell viability:. Application to the quantitation of cytotoxic and growth inhibitory lympolines J . Immuno. methods 1984, 70, 257-268.) the 3- (4,5-dimethylthiazol-2- yl ) -2,5-diphenyl tetrazolium bromide (MTT) assay was performed according to the method of. The cultured cells were counted by 0.4% trypan blue staining, and then dispensed into each well at 5 로 10 ⁴ ce11s / 200 μL in 96 well plates. After 24 hours of incubation, the medium was removed and in 200 μL of fresh DMEM medium. The dissolved concentration samples were added to each well and incubated for 24 hours, followed by LPS (1 μg / mL) for 24 hours. 10 μL of MTT (5 mg / mL) solution was added to each well and the culture medium to which LPS was added, followed by incubation for 4 hours. After incubation, the supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the formazan crystals, and the absorbance was measured at 550 nm with an ELISA reader.

1.4.3 Nitric oxide (NO) production measurement

The degree of NO production was determined by Green et al. (Green, LC; Wagner, DA; Glogowski, J. Analysis of nitrate, and nitrate in biological fluids.Anal . Biochem. 1982 , 126 , 131-138.). It was measured using the amount of NO ₂ ⁻ generated in the medium. After dispensing RAW 264.7 cells into a 24 well plate at a concentration of 1 ce10 ⁵ ce11s / mL using DMEM, the samples were incubated for 24 hours by concentration, and then incubated again by adding LPS (1 μg / mL) for 24 hours. I was. 100 μL of the cell culture supernatant and 100 μL of Griess reagent (1% sulfanilamide, 0.1% naphthylethylendiamine in 2.5% phosphoric acid) were mixed for 10 minutes in 96 well plates, and the absorbance was measured at 540 nm using an ELISA reader. NO ₂ ^-The standard curve was used to prepare NaNO ₂ by concentration.

1.4.4 ProstaglandinE ₂ (PGE ₂ ) Measure

After dispensing RAW 264.7 cells into a 24 well plate at a concentration of 1 ce10 ⁵ ce11s / mL using DMEM, the samples were incubated for 24 hours by concentration, and then incubated again by adding LPS (1 μg / mL) for 24 hours. I was. PGE ₂ production was measured by the supernatant. Antibodies were precoated overnight with coating buffer, washed with 0.01 M PBS containing 0.05% Tween-20 and incubated in 96 well plates with blocking buffer. After removing the blocking buffer, the sample was added and then cultured again. Secondary antibodies were attached to the washed 96 well plates, and then absorbance was measured at 450 nm by adding a detection reagent, a substrate, and a stopping reagent.

1.4.5 TNF-α Measure

After dispensing RAW 264.7 cells into a 24 well plate at a concentration of 1 ce10 ⁵ ce11s / mL using DMEM, the samples were incubated for 24 hours by concentration, and then incubated again by adding LPS (1 μg / mL) for 24 hours. I was. The amount of TNF-α production was measured with the supernatant. Antibodies were precoated overnight with coating buffer, washed with 0.01 M PBS containing 0.05% Tween-20 and incubated in 96 well plates with blocking buffer. Blocking buffer was removed, sample was added, and then incubated again. The secondary antibody was attached to the washed 96 well plates, and the absorbance was measured at 450 nm by adding a detection reagent, a substrate, and a stopping reagent.

1.5 Expression of Atherosclerosis-related Genes

1.5.1 Gene Expression Survey by RT-PCR Method

RNA was incubated for 24 hours in 4 mL of medium by splitting RAW 264.7 cells (1 ㅧ 10 ⁶ cells / mL) into a D60 culture dish, and then adding Molokia methanol extract and its fractions by concentration, and again 1 μg / mL. 1 μL of LPS was added and incubated for 24 hours. To remove RNA, the media of D60 was removed, washed three times with 1 mL of PBS (pH 7.4), and treated with TRI reagent. Add 1 mL of this to a sterile 1.5 mL tube, add 200 μL of 1-bromo-3-chloropropane, vortex for 30 seconds, leave on ice until the layers separate, and centrifuge (15,000 rpm, 20 minutes, 4 ℃). The supernatant was taken and transferred to a new tube. The same amount of isopropanol was added thereto, left at -20 ° C for at least 2 hours, centrifuged (15,000 rpm, 20 minutes, 4 ° C), and the supernatant was removed. 75% ethanol was added to the precipitate, and then distilled water treated with diethyl pyrocarbonate (DEPC) was added thereto to react for 10 minutes in a 65 ° C. water bath to dissolve the precipitate. The isolated RNA was identified and quantified by UV / visible spectrophotometer and 2% agarose gel electrophoresis.

The total RNA 2 μg separated, RNA PCR kit was mixed and reacted at 42 ℃ for 90 minutes and then heated at 70 ℃ for 10 minutes to terminate the reaction.

CDNA generated by reverse transcription of total RNA was amplified by PCR using the primers of Table 2 below. PCR reaction was performed under the conditions for each primer by adding 2.5 U Taq polymerase (Takara Ex.), 2 mM MgCl ₂ and 0.2 mM dNTP. As an amplification profile, the initial step was performed at 94 ° C for 2 minutes and 30 seconds, followed by denaturation at 95 ° C for 40 seconds, annealing at each annealing temperature for 1 minute and 20 seconds, and 30 cycles of polymerization for 2 minutes at 72 ° C. The reaction was further extended for 10 minutes at and stored at 5 ° C. The primers used here are ICAM-1, VCAM-1, MCP-1, iNOS, COX-2, GAPDH and the like, as shown in Table 2 below.

Gene Fragment size (bp) Oligonucleotides ICAM-1 170 F 5 ㅄ -AAC TCT TCC AGA ACA CCT CG-3 ㅄ R 5 ㅄ -AGC ACC AGC TAG ACC TTA GC-3 ㅄ VCAM-1 206 F 5 ㅄ -TCA TCC CCA CCA CTG AAG AT-3 ㅄ R 5 ㅄ -GCC ATT GCT AGA GCA TGT CA-3 ㅄ MCP-1 267 F 5 ㅄ -GTG CCT GCT ACT CAC AGT AG-3 ㅄ R 5 ㅄ -GGA ATT TGG TTT TTC TTG TT-3 ㅄ iNOS 920 F 5 ㅄ -GCC TTC AAC ACC AAG GTT GTC TGC A-3 ㅄ R 5 ㅄ -TCA TTG TAC TCT GAG GGC TGA CAC A-3 ㅄ COX-2 490 F 5 ㅄ -TGG GCA AAG ATG CAA ACA TC-3 ㅄ R 5 ㅄ -CAG CAA ATC CTT GCT GTT CC-3 ㅄ GAPDH 514 F 5 ㅄ -GGA AGG CCA TGC CAG TGA-3 ㅄ R 5 ㅄ -TGA GAA CGG GAA GCT TGT CA-3 ㅄ

1.5.2. Investigation of protein expression by Western blot

Western blot was performed to investigate the effects of Molokia methanol extract and its fractions on protein expression of inflammation-related gene regulators NFκB and IL-1β. After incubation in RAW 264.7 cells (1mL10 ⁶ cells / mL) for 24 hours using a DMEM medium in a 5% CO ₂ incubator, the medium was removed and cultured under the same conditions as the pre-culture. After washing the cells with PBS 2-3 times, 1 mL of lysis buffer was added, dissolved for 30 minutes to 1 hour, and centrifuged at 13,000 rpm for 10 minutes to remove cell membrane components. Protein concentration was quantified using the Bio-Rad Protein Assay Kit standardized to bovine serum albumin (BSA). The supernatant centrifuged at 13,000 rpm for 10 minutes at 4 ℃ was subjected to SDS-polyacrylamide gel electrophoresis at 125 V using a 10% running gel and 4.5% stacking gel.

Proteins isolated by electrophoresis were transferred for 120 minutes at 350 mA using immobilon-P transfer membrane and transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine). The protein-translated membrane was blocked with 5% non-fat skim milk solution after confirming the transfer to the fast green solution. In order to examine the expression levels of primary antibodies NFκB and IL-1β at 4 ° C, anti-rabbit and anti-mouse were diluted 1: 1000 in TTBS solution for 24 hours, and then washed three times with TTBS. As a secondary antibody, HRP (Horse Radish Peroxidase) conjugated anti-mouse or anti-rabbit IgG was diluted 1: 5000 and reacted for 2 hours, followed by TST (100 mM Tris-HCl, 1.5 M NaCl, 0.5% tween-). 20) was washed three times at 10 minute intervals. To the membrane reacted with the antibody, a mixed solution containing 40: 1 of the colorants I and II of the ECL diagnostic kit was added.

1.6. Statistical analysis

The experimental results were analyzed by ANOVA using SAS program (SAS Institute, Inc. SAS / SAAT user's Guide.version 6, 4th ed.Cary, NC, USA. 1998.) and then Duncan's multiple range test for significant differences. Significant differences between the samples were tested at the p <0.05 level.

2. Results and Discussion

2.1 Composition of Molokia

2.1.1 General Constituents of Molokia

The results of analyzing the general components of the Molochia powder are shown in Table 3 below. Carbohydrate 55%, crude fat 2.5%, crude protein 17%, moisture 6.1%, crude ash 12.9%, crude fiber 6.5%.

Constituent Content (%) Moisture  6.1 Carbohydrate 55.0 Crude fat 2.5 Crude protein 17.0 Crude ash 12.9 Crude fiber 6.5

  2.1.2 Systematic Fraction Yield of Molokia Extract

The yield of the methanol extract of Molochia was sequentially divided according to polarity with n- hexane, chloroform, ethylacetate, n- butanol and water as shown in Table 4 below.

Weight (g) of fraction recovered (%) Extract Fractions Methanol n -Hexane Chlororoform Ethylacetate n -Butanol Water 25.0 2.1 1.2 16.1 27.4 45.6

Percentage of each fractions to methanol extract content (100 g)

2.2 Antioxidant Effect of Molokia

  2.2.1 Total Polyphenol Content and Total Flavonoid Content

  The total polyphenol and flavonoid contents in the molokia extracts and fractions were compared and measured using tannic acid and quercetin as reference materials, and the results are shown in Table 5 below. As a result, the polyphenol content was very high at 295.03, 108.53 and 103.54 μg / mg in ethylacetate, chloroform and butanol fractions, respectively. The total flavonoid content was the highest in the ethylacetate fraction of 230.92 μg / mg.

Sample Total phenolics ¹⁾ (μg / g) Total flavonoids ²⁾ (μg / g) Methanol ext. 79.81 ± 8.88 35.93 ± 0.46 n -Hexane fr. 55.88 ± 1.65 55.82 ± 0.46 Chloroform fr. 108.53 ± 9.10 50.05 ± 1.04 Ethylacetate fr. 295.03 ± 32.00 230.92 ± 13.47 n -Butanol fr. 103.54 ± 12.27 29.78 ± 7.33 Water fr. 34.29 ± 5.51 4.93 ± 0.46

¹⁾ Total phenolics is expressed as tannic acid equivalents.

²⁾ Total flavonoids is expressed as quercetin equivalents.

  2.2.2 DPPH radical scavenging activity

DPPH is a relatively stable free radical with a deep purple color, which is reduced and decolorized by sulfuric acid amino acids such as cysteine and glutathione, ascorbic acid, and aromatic amines. ROS is mostly eliminated by the body's defense mechanisms, but when it is not removed, it reacts rapidly with biomolecules, causing protein denaturation, biofilm lipid peroxidation, DNA damage, and the like. Lipid peroxides that diffuse into cells or migrate through the bloodstream are new radicals. It promotes the reaction and acts as a cause of various chronic diseases and aging.

The results of comparing the DPPH scavenging activity of the Molochia extract and fractions with butylated hydroxyanisole (BHA) by concentration are shown in FIG. 3. The antioxidant activity was about 90% at 25 μg / mL of BHA used as a standard, and the scavenging activity was about 94% at 200 μg / mL of each of the Molokia ethylacetate, chloroform, butanol and methanol extracts. . The RC ₅₀ values of ethylacetate and butanol fractions were 17.22 μg / mL and 57.95 μg / mL, respectively.

In Figure 3, M: methanol extract, H: n- hexane fraction, C: chloroform fraction, E: ethylacetate fraction, B: n -butanol fraction, W: water fraction.

  2.2.3 ABTS radical scavenging activity

ABTS + · produced by leaving ABTS and potassium persulfate in cows was eliminated by the antioxidant power of the extract, and the ABTS + · scavenging ability of the extract can be observed by measuring the degree of decolorization of the index-specific blue-green color. The ABTS radical scavenging method, developed on the basis of the fact that the absorbance of ABTS cationic radicals in plasma is inhibited by antioxidants, can be compared with the standard value of Trolox and can be used to measure antioxidant activity in vivo as well as in vitro . It is widely used as a method for measuring.

  As a result of measuring the ABTS + scavenging activity of the Molochia extract and fractions compared to Trolox, as shown in FIG. 4, the Molokia ethylacetate fraction, the chloroform fraction, butanol fraction and methanol extract were superior to 90% at the concentration of 100 μg / mL. The scavenging activity was shown to be greater than 90% for the molokia ethylacetate fraction and over 80% for the chloroform fraction at the treatment concentration of 50 μg / mL, and 60 ~ 70 for the butanol fraction and the methanol extract. There was a scavenging activity of%. However, molochia heaxane and water fractions showed scavenging activity at concentrations of 100 μg / mL or more. In particular, the RC50 values of the molokia ethylacetate fraction and chloroform fraction with excellent ABTS + and scavenging activity were 7.42 μg / mL and 30.04 μg / mL, respectively. The results showed that the extracts and fractions of Molokia showed superior ABTS + and scavenging activity, similar to those showing excellent DPPH scavenging activity.

In Figure 4, M: methanol extract, H: n- hexane fraction, C: chloroform fraction, E: ethylacetate fraction, B: n -butanol fraction, W: water fraction, but * H, W: 100, 250 , 500 μg / m, Trolox: 15 μM concentration.

This is summarized in Table 6 below.

Sample RC ₅₀ ¹⁾ (μg / mL) DPPH ABTS Methanol ext. 58.42 ± 4.95 46.93 ± 3.15 n -Hexane fr.    170.69 ± 11.23 69.63 ± 2.71 Chloroform fr. 70.60 ± 6.70 30.04 ± 0.86 Ethylacetate fr. 17.22 ± 2.21 7.42 ± 0.62 n -Butanol fr. 57.95 ± 1.25 36.02 ± 1.40 Water fr. 152.14 ± 16.42 144.41 ± 8.22

¹⁾ Concentration required for 50% reduction of DPPH and ABTS + · at 1 min after starting the reaction

²⁾ Each value represent the mean ± SD from 3 independent experiments

* p <0.05

  2.2.4 Component Determination of Polyphenolic Compounds

  To determine the changes of chlorogenic acid, quercetin-3 -glucoside, quercetin-3-galactoside, and quercetin in the polyphenolic compounds in Molokia, the content of Molokia extracts and fractions was analyzed using HPLC. It is shown in Table 7 below. In the following Table 7, the unit is mg / 100 g.

 Compound Methanol ext. Ethylacetate fr. Hexane fr. Chloroform fr.  Chlorogenic acid 646.5 203.2 2.1 1.1  Quercetin-3-glucoside 70.21 175.2 0.7 0.4 Quercetin-3-galactoside 82.15 216.6 0.8 0.7  Quercetin 22.34 0 2.0 0.2

 As a result, the extract of Molochia was 646.5 mg of chlorogenic acid, 70.2 mg of quercetin-3-glucoside, 82.15 mg of quercetin-3-galactoside, and 22.3 mg / 100 g of quercetin.

In particular, the ethylacetate fraction showed the highest content of polyphenolic compounds (203.2 mg of chlorogenic acid, 175.2 mg of quercetin-3 -glucoside, and 216.6 mg / 100 g of quercetin-3-galactoside). But The hexane and chloroform fractions contained trace amounts of polyphenol compounds.

  In the ethylacetate fraction, the highest content of quercetin-3-galactoside was found in the polyphenolic compounds, and the highest content of chlorogenic acid in methanol extracts.

Figure 5 shows the chemical structure of phenolic antioxidants isolated from Molokia.

Meanwhile, FIG. 6 shows an HPLC chromatogram of the Molokia methanol extract. In FIG. 6, 1: chlorogenic acid, 2: quercetin-3-glucoside 3: quercetin-3-galactoside, and 4: quercetin are shown.

2.3 Anti-inflammatory Effects of Molokia Extract

  2.3.1 Cell survival rate

Lipopolysaccharide (LPS) is a cell wall material of Gram-negative bacteria, which has been reported to stimulate immune cells to increase NO production. In the RAW 264.7 cells induced oxidative stress with LPS, the cell survival is shown in Fig. 7 when the Morocea extract and fractions were treated by concentrations and cultured for a predetermined time. Survival rate of RAW 264.7 cells stimulated by LPS was reduced compared to non-LPS-treated controls, but when treated with 25-250 μg / mL of the Molochia extract and fraction concentrations, the other treatments except for butanol fractions were more than 50% cells. Survival rate was shown. At the highest concentration of 500 μg / mL, however, cell viability of 50-68% was observed only in Molokia methanol extract and ethylacetate fraction.

In Figure 7, M: methanol extract, H: n- hexane fraction, C: chloroform fraction, E: ethylacetate fraction, B: n -butanol fraction, LPS: alone, Con: control.

  2.3.2 NO Formation Conditions and Amount

In order to determine the concentration and time of LPS treatment required to induce oxidative stress in RAW 264.7 cells, we measured NO production according to LPS treatment concentration and culture time and observed the effect on NO production of Molokia extract. Is shown in FIG. 8. When LPS concentration was 0 ~ 6 μg / mL and treatment time was 6, 12, 18, 24, 48 hr, NO production increased by 10 times when LPS treatment concentration was over 1 μg / mL. Was not. In addition, the NO production amount was increased by varying the incubation time by treating LPS with 1 μg / mL, and the production amount of NO was increased by about 10 times at 24 hr. Therefore, in this experiment, LPS treatment concentration was determined to be 1 μg / mL and incubation time to 24 hr.

The LPS treatment increased NO production than the control without LPS, which is consistent with the report that LPS causes oxidative stress. When intracellular oxidative stress was induced by LPS, NO production was significantly increased, and NO production was inhibited when Molokia extract and fractions were added. The results seen in moles Escherichia extract and fractions is the superoxide anion generation by the NADPH oxidase to inhibit the production of NO in macrophages during the initial inflammatory reaction (O ₂ ^-) and the peroxynitrite (ONOO ^-) a strong oxidant in the reaction of NO May inhibit cytotoxicity and LDL oxidation.

  In normal physiological state, NO mediates important physiological functions such as blood vessel homeostasis, but in hyperlipidemia and atherosclerosis state, oxidative stress is induced by peroxynitrite produced by the reaction of excess superoxide and NO. In response, it impairs the function of proteins or stimulates the expression of genes for cell protection, leading to cell damage or cell death.

At 50 μg / mL of each of hexane, chloroform and ethylacetate fractions of Molochia, about 50% of NO production was inhibited. In particular, ethylacetate fraction showed a high inhibitory effect of more than 50%, which is illustrated in FIG. 9. Therefore, the molokia ethylacetate fraction seems to have the highest NO inhibitory activity, which may be related to the ethylacetate fraction of the molokia fraction containing the most polyphenols.

9, M: methanol extract, H: n- hexane fraction, C: chloroform fraction, E: ethylacetate fraction, B: n- butanol fraction, LPS: alone, and Con: control.

  2.3.3 PGE ₂ Suppress generation

NO is known to promote PGE ₂ biosynthesis and intensify inflammation. In addition, many of the iNOS inhibitors isolated from natural products have both COX-2 inhibitory activity and PGE ₂ inhibitory effect, and have been reported to inhibit the expression of COX-2 and iNOS by regulating the transcription factor NFκB. .

It was confirmed that the Molochia extract and fraction inhibited the production of PGE ₂ in RAW 264.7 cells treated with LPS. As shown in FIG. 10, PGE ₂ production was inhibited by about 50% when 100 μg / mL of hexane, chloroform, and ethylacetate fractions were treated. In particular, ethylacetate fractions had an inhibitory activity of 50% or more even at a concentration of 50 μg / mL. Showed the greatest inhibitory activity. Therefore, it is thought that Molochia fraction can inhibit or reduce inflammation by inhibiting PGE ₂ production.

In FIG. 10, M: methanol extract, H: n- hexane fraction, C: chloroform fraction, E: ethylacetate fraction, LPS: alone, and Con: control.

  2.3.4 Inhibition of TNF-α Production

  TNF-α, a type of inflammatory cytokine, is known as a factor that induces atherosclerosis by activating NFκB of vascular endothelial cells to bind to adhesion materials such as VCAM and ICAM.

  The inhibitory activity of TNF-α production was confirmed in RAW 264.7 cells stimulated with Molokia extract and fractions. As shown in FIG. 11, TNF-α increased by LPS was inhibited in inverse proportion to the addition concentrations of Molokia methanol extract and hexane, chloroform, and ethylacetate fractions. In particular, the production of TNF-α was significantly inhibited in the chloroform and ethylacetate fractions. It became. In the case of the Molochia ethylacetate fraction, more than 40% of TNF-α production was inhibited even at 25 μg / mL treatment concentration, showing the highest inhibitory activity of TNF-α production.

In Figure 11 M: methanol extract, H: n- hexane fraction. C: chloroform fraction, E: ethylacetate fraction, LPS: alone, and Con: control.

  2.3.5 Effects on the Expression of Atherosclerosis-related Genes

   2.3.5.1 VCAM-1, ICAM-1 Expression

   To observe the effect of Molokia extract on the production of adhesion molecules in RAW 264.7 cells induced oxidative stress with LPS, RT-PCR expression of soluble VCAM-1 and ICAM-1 involved in the early development of atherosclerosis It confirmed by the method and the result is shown in FIG. When the Molochia methanol extract was treated by concentration, the expression of VCAM-1 and ICAM-1 decreased with the increase of treatment concentration. However, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) selected to standardize the amount of mRNA used to express these genes showed no difference according to the concentration of sample treatment, indicating that LPS treatment did not affect target gene expression. there was.

In the early stages of inflammation, the expression of cell-adhesive substances such as VCAM-1 and ICAM-1 increases on the surface of endothelial cells, attracting leukocytes and monocytes circulating in the blood to deepen the atherosclerosis and Inflammatory cytokine and IL-1 increase the expression of cell adhesion substances in the vascular endothelium. Therefore, Methanol extract of Molochia is thought to be able to effectively inhibit the early development of atherosclerosis by significantly reducing the gene expression of cell adhesion material.

   2.3.5.2 MCP-1, iNOS, COX-2 Expression

  Recently, studies on polyphenol compounds that inhibit NFκB activity that modulates cytokine and LPS-induced iNOS expression in macrophages and chondrocytes are very active. Therefore, monocyte chemotactic protein-1 (MCP-1) and inducible NOS (iNOS) in RAW 264.7 cells treated with quercetin, chlorogenic acid, quercetin-3-galactoside, quercetin-3-glucoside and LPS ) And the effect on COX-2 gene expression were confirmed by RT-PCR method.

   MCP-1 is known to be expressed mainly by macrophages in the early stages of atherosclerosis lesions and is also expressed by smooth muscle. In Molochia methanol extract, MCP-1 gene expression was reduced above 100 μg / mL, and iNOS significantly decreased at 500 μg / mL. This is shown in FIG. 13.

   In addition, the treatment concentration for each molokia polyphenol compound was performed by setting the concentration without toxicity based on the cell viability test results. Gene expression in iNOS showed that quercetin decreased iNOS gene expression at 25 μg / mL, chlorogenic acid was not changed, 10 μg / mL at quercetin-3-galactoside, and 5 at quercetin-3-glucoside. iNOS gene expression decreased over μg / mL. The results are shown in FIG.

In COX-2 expression, gene expression was decreased at 25 μg / mL of quercetin and chlorogenic acid, respectively, and quercetin-3-galactoside was significantly decreased at 10 μg / mL. In contrast, quercetin-3-glucoside had no effect on COX-2 gene expression. The results are shown in FIG.

   INOS, a NO production inducing enzyme involved in the regulation of vascular relaxation, platelet aggregation and cardiovascular homeostasis, was also treated with quercetin as shown in FIG. , quercetin-3-galactoside and quercetin-3-glucoside.

   In Fig. 14 1: control, 2: LPS: alone, Q (3-5): 5, 15, 25 μg, CA (3-5): 10, 25, 50 μg, Qgc (3-5): 1, 5, 10 μg Qgu (3-5): 1, 5, 10 μg, Q: Quercetin, CA: Chlorogenic acid, Qgc: Quercetin-3-galactoside, Qgu: Quercetin-3-glucoside.

   iNOS does not normally exist intracellularly, but when induced, it produces a large amount of NO for a long time, and NO produced by iNOS in inflammatory state not only promotes inflammatory reactions such as vascular permeability and edema, but also promotes biosynthesis of inflammatory mediators. It is known to deepen inflammation. NO is involved in the development of inflammation and cancer, and iNOS expression is induced by NFκB activity, which is an important mechanism by which LPS or cytokine is overproduced by macrophage. Several natural antioxidants, polyphenol compounds, are known to inhibit inflammation by directly inhibiting the expression of NFκB-dependent cytokine and iNOS genes. Induction of NO production using LPS (1 μg / mL) in RAW 264.7 cells and iNOS gene expression decreased as the treatment concentration was increased by the concentration of Molochia extract and polyphenol compounds. Inhibition of activity and the anti-Molochia polyphenol compounds are expected to have an anti-inflammatory effect.

COX is an enzyme that converts arachidonic acid to prostaglandinE ₂ (PGE ₂ ). It is classified as COX-1 and COX-2. COX-1 acts on normal biological functions such as platelet formation, gastric wall protection, and renal function maintenance. The mechanism of action of many anti-inflammatory drugs suggests inhibition of prostaglandin synthesis, which is due to the production and deactivation of COX-2. Induction of COX-2 production by using LPS (1 μg / mL) in RAW 264.7 cells was confirmed that gene expression was inhibited as the treatment concentration increased in these samples when treated with concentrations of molokia polyphenol compounds. The results are shown in FIG. 15.

   15: 1, control, 2: LPS: alone, Q (3-5): 5, 15, 25 μg, CA (3-5): 10, 25, 50 μg, Qgc (3-5): 1, 5, 10 μg, Qgu (3-5): 1, 5, 10 μg, Q: Quercetin, CA: Chlorogenic acid, Qgc: Quercetin-3-galactoside, Qgu: Quercetin-3-glucoside.

Therefore, the synthesis of PGE ₂ by COX-2 is thought to promote the inflammatory response, and it was confirmed that Molochia antioxidants that inhibit the production and activity of COX-2 have an anti-inflammatory effect.

Based on the above results, Molochia extract and polyphenolic compounds of Molokia seem to be able to act as anti-inflammatory agents by inhibiting the mRNA expression of MCP-1, iNOS, COX-2 increased by LPS.

    2.3.5.3 NFκB Protein Expression

    NFκB is a transcription factor involved in various stages such as cytokine response, inflammation, and cell growth regulation. Recent studies strongly suggest that inducible NFκB is involved in the development of atherosclerosis. In many cells, NFκB is a redox-sensitive transcription factor that is involved in various signal transduction from the cytoplasm to the nucleus, and is present as a trimer of p50, p65, and IκB subunits in the cytoplasm. When IκB is degraded by oxidative stress, p50, p65 heterodimer Is known to migrate into the nucleus resulting in continuous DNA binding.

  Molecular extracts and fractions, and the effects of quercertin, chlorogenic acid, quercetin-3-galactoside and quercetin-3-glucoside on LPS-induced oxidative stress in RAW 264.7 cells were investigated. As a result, NPSκB activity was induced only by treatment with LPS, but NFκB activity was inhibited as the treatment concentration increased compared to the LPS treatment when the Molochia methanol extract was treated. At the concentration of 250 μg / mL of extract of Molokia and 100 μg / mL of ethylacetate fraction, NFκB activity was reduced by about three times compared to that of LPS treatment, and NFκB activity was most inhibited at 100 μg / mL of ethylacetate fraction. It is shown in FIG. In addition, the cell viability was measured at a concentration of 25 μg / mL quercertin, 50 μg / mL chlorogenic acid, 10 μg / mL quercetin-3-galactoside, and 10 μg / mL quercetin-3-glucoside. In these treatments, NFκB activity was reduced by about four times as compared to LPS treatment, which is shown in FIG. 18.

  From these results, it was confirmed that Mollocia methanol extract, ethylacetate fraction and polyphenol compounds such as quercertin, chlorogenic acid, quercetin-3-galactoside and quercetin-3-glucoside also inhibited NFκB activity, a transcription factor of oxidative stress or inflammation. have.

In FIG. 17, M250: methanol extract 250 μg / mL, E100: ethylacetate fraction 100 μg / mL, C50: chloroform fraction 50 μg / mL, and H50: hexane fraction 50 μg / mL.

In Fig. 18, Q25: 25 μg / mL of quercetin, CA50: 50 μg / mL of chlorogenic acid, Qgc10: 10 μg / mL of quercetin-3-galactoside, and 10 μg / mL of Qgu10: quercetin-3-glucoside.

    2.3.5.4 Protein Expression of IL-1β

    IL-1β works in conjunction with TNF-α to act as an inflammatory cytokine in the immune response in the presence of infection or tumors. In addition, TNF-α stimulates the production of adhesion substances such as ICAM-1 and VCAM-1 by stimulating endothelial cells, which are structures between blood and tissues, and move various leukocytes to inflammatory sites. Through these mechanisms, white blood cells accumulate in the inflammatory site, which constantly secrete various cytokines to maintain toxicity to microorganisms and tumor cells, and induce inflammatory reactions to damage tissues.

    Effects of Molokia Extracts and Fractions and Polyphenol Compounds quercertin, Chlorogenic Acid, Quercetin-3-galactoside, and Quercetin-3-glucoside on Interleukine-β (IL-1β) Activity in RAW 264.7 Cells Induced Oxidative Stress with LPS The effect was observed. As a result, the Molokia methanol extract was significantly inhibited IL-1β activity as the treatment concentration increases compared to the LPS treatment, the results are shown in Figure 19. At the concentration of 100 μg / mL of ethylacetate fraction and 50 μg / mL of hexane fraction, IL-1β activity was reduced by 3 times compared to LPS treatment. Especially, 100 μg / mL of ethylacetate fraction showed the greatest inhibition of IL-1β activity. The result is illustrated in FIG. 20.

    In addition, chlorogenic acid and quercetin were treated with quercetin 25 μg / mL chlorogenic acid 50 μg / mL quercetin-3-galactoside 10 μg / mL and quercetin-3-glucoside 10 μg / mL. In the -3-galactoside treatment, IL-1β activity was decreased by about three times, and the treatment of quercetin and quercetin-3-glucoside was inhibited by about two times, and the result is shown in FIG. 21. It was.

As such, Molokia methanol extract, ethylacetate fraction and quercetin, chlorogenic acid, quercetin-3-galactoside, quercetin-3-glucoside also block NO production by blocking NFκB activity, a transcriptional step of IL-1β-induced iNOS expression in RAW 264.7 cells. By inhibiting, it can be seen that the polyphenolic compound of molokia inhibits IL-1β activity.

Meanwhile, in FIG. 20, M250: methanol extract 250 μg / mL, E100: ethylacetate fraction 100 μg / mL, C50: chloroform fraction 50 μg / mL, H50: n- hexane fraction 50 μg / mL.

In Fig. 21, Q25: 25 μg / mL of quercetin, CA50: 50 μg / mL of chlorogenic acid, Qgc10: 10 μg / mL of quercetin-3-galactoside, and 10 μg / mL of Qgu10: quercetin-3-glucoside.

On the other hand, foods to which the extract may be added include, for example, various foods, meats, beverages, chocolates, snacks, confections, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic beverages, vitamin complexes, and health supplements. Foods, etc., but is not limited thereto.

When applied to foods like this, it is possible to easily consume the molochia extract all the time, from a wide range of adults and the elderly to expect a continuous drinking effect.

In the present invention, when the ethyl acetate fraction of the molokia extract, particularly the molokia extract having excellent activity, is applied to food, it is preferable to contain at least 0.01% by weight or more based on the total composition in terms of its effectiveness. In the case of Molokia extract, even if the content is included in excess, it does not show toxicity, so it is not particularly meaningful to set an upper limit of the added amount.

An example of manufacturing into food may be as follows, but each food composition is not limited.

(1) Preparation of functional drinks

A composition containing a general functional beverage base comprising 0.01% by weight ethyl acetate fraction, Methanol 0.05%, orange essence 0.05%, excess 7.0%, citric acid 0.1%, and vitamin C 0.05% by weight of Molokia methanol extract To prepare a beverage by adding purified water.

(2) the manufacture of medicine

The deodorized purified 40% by weight of alcohol is diluted with distilled water, 0.01% by weight of ethyl acetate fraction of the Molochia methanol extract is added, and stevioside, high fructose, amino acid, citric acid, salt and the like are added to the alcohol content concentration of 15 to 30. Made by weight percent.

(3) manufacture of healthy foods

1,000 mg ethylacetate fraction of Molokia methanol extract, a suitable vitamin mixture (70 μg vitamin A acetate, 1.0 mg vitamin E, 0.13 mg vitamin B1, 0.15 mg vitamin B2, 0.5 mg vitamin B6, 0.2 μg vitamin B12, vitamin C 10 mg) ), 10 μg biotin, 1.7 mg nicotinic acid amide, 50 μg folic acid, 0.5 mg calcium pantothenate, an appropriate amount of inorganic mixture (1.75 mg ferrous sulfate, 0.82 mg zinc carbonate, 25.3 mg magnesium carbonate, 15 mg potassium phosphate monobasic 55 mg of calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, 24.8 mg of magnesium chloride) are mixed, and then granules are prepared and used for preparing a health food composition according to a conventional method.

1 is a schematic of possible active sites of antioxidants.

2 is the main classification of polyphenols.

Figure 3 shows the DPPH radical scavenging activity of butylated hydroxyanisole (BHA), Molokia methanol extract and fractions.

4 shows ABTS radical scavenging activity of Trolox, Molokia methanol extracts and fractions.

5 is the chemical structure of a phenolic antioxidant isolated from Molokia.

Figure 6 HPLC chromatograms of Molokia methanol extract and fractions.

7 is an effect on the proliferation of RAW 264.7 cells of the methanol extract and fractions Molokia.

FIG. 8 is a graph showing the effect of LPS concentration on NO production in RAW 264.7 cells (A) and the effect of constant temperature (B). FIG.

FIG. 9 is a graph showing the effect of Molokia methanol extract and fractions on NO production in LPS-treated RAW 264.7 cells.

10 is a graph showing the effect of Molokia methanol extract and fractions on LPS-induced PGE ₂ production.

FIG. 11 is a graph showing the effect of Molokia methanol extract and fractions on LPS-induced TNF-α production in RAW 264.7 cells.

12 is a graph showing the VCAM-1 inhibitory effect (A) and ICAM-1 inhibitory effect (B) of Molokia methanol extract in LPS-induced RAW 264.7 cells.

Figure 13 is a graph showing the MCP-1 inhibitory effect (A) and iNOS inhibitory effect (B) of Molokia methanol extract in LPS-induced RAW 264.7 cells.

FIG. 14 is a graph showing the iNOS inhibitory effects of molokia compounds in LPS-induced RAW 264.7 cells. FIG.

FIG. 15 is a graph showing the effect of COX-2 inhibition of Molochia compounds in LPS-induced RAW 264.7 cells. FIG.

16 is a graph showing the effect of Molokia methanol extract on NF-κB production in LPS-induced RAW 264.7 cells.

FIG. 17 is a graph showing the effect of Molokia fractions on NF-κB production in LPS-induced RAW 264.7 cells.

FIG. 18 is a graph showing the NF-κB inhibitory effect of Molochia compounds in LPS-induced RAW 264.7 cells. FIG.

19 is a graph showing the effect of Molokia methanol extract on IL-1β production in LPS-induced RAW 264.7 cells.

FIG. 20 is a graph showing the effect of the molokia fractions on IL-1β production in LPS-induced RAW 264.7 cells.

FIG. 21 is a graph showing the IL-1β inhibitory effect of the molokia compounds in LPS-induced RAW 264.7 cells. FIG.

Claims (6)

Food with anti-inflammatory activity, including Molokia methanol extract. A food having anti-inflammatory activity comprising an ethyl acetate fraction of Molokia methanol extract. A food having anti-inflammatory activity containing quercetin-3-galactoside derived from Molokia. The food according to any one of claims 1 to 3, which inhibits nitric oxide (NO), PGE ₂ (Prostagladin E ₂ ), and Tumor neroses factor-1 (TNF-α) production. The method according to any one of claims 1 to 3, characterized by inhibiting mRNA gene expression of Monocyte chemotactic protein-1 (MCP-1), Inducible NOS (iNOS), Cyclooxygenase-2 (COX-2). food. The food according to any one of claims 1 to 3, which inhibits protein expression of Cyclooxygenase-2 (COX-2), Nuclear fator κB (NFκB) and Interleukine-1β (IL-1β).

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